Sodium 4-(4-substitutedanilino)-4-substituedbut-2-enoate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Jun - 10 Jul 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Testing Methods for New Chemical Substances

Version / remarks:

(March 31, 2011, No.0331-7, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; March 29, 2011, No.5, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 110331009, Environmental Policy Bureau, Ministry of the Environment, Japan.) IV. Algae growth inhibition test

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, 1.0; 3.2; 10; 32; 100 mg/L
- Sampling method: At initiation of exposure: an aliquot of remaining unused test solution of each test group was used for measurement. At the termination of exposure: the same quantity of the sample taken from each replicate was mixed and analyzed per each test group. The test solution was centrifuged (2000 g, 10 min, 22 °C) and the aqueous layer without algae was analyzed for test substance concentration (n = 1).

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 0.1183 g of the test substance was weighed with the electronic balance. A stock solution was prepared by mixing the test substance with the medium to 100 mL and then stirred for 48 h with a magnetic stirrer at 22 ± 2 °C. Each test solution was prepared by diluting an appropriate volume of the stock solution with the medium to a volume of 300 mL.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: ATCC 22662
- Source: American Type Culture Collection (date received: 01 Aug 1996)
- Age of inoculum (at test initiation): 3 d

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

21.3 - 23.7 ° C

pH:

7.6 - 7.8 at initiation of exposure
7.8 - 7.9 at termination of exposure

Nominal and measured concentrations:

nominal: control, 1.0, 3.2, 10, 32, 100 mg/L
mean measured concentration: control, 1.04, 3.34, 10.4, 33.5, 104 mg/L

Details on test conditions:

TEST SYSTEM
- Type: closed with an air-permeable silicone stopper
- Material, size, headspace, fill volume: glass; 300 mL; headspace: 250 mL; fill volume: 50 mL
- Initial cell density: 0.5 x 1E+04 cells/mL
- Control end cell density: Mean = 557500 cells/mL, S.D. = 62659 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous fluorescent illumination: cool-white light
- Light intensity and quality: Wave length: 400 - 700 nm; Light intensity: 60 - 120 μE/m²/s ± 15% (at the surface of the cultures)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: electronic particle counter at 24, 48 and 72 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: yes
- Test concentrations: 1.0, 3.2, 10, 32 and 100 mg/L of test substance
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 104 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.34 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: yes
- Colour differences: test solution was slighty yellow at nominal concentrations of 32 and 100 mg/L

Results with reference substance (positive control):

- Results with reference substance valid? yes
- ErC50 (0 - 72 h): 1.14 mg/L
- Historical ErC50 (0 - 72 h): 1.1 mg/L ± 0.26 mg/L (Mean ± S.D.)

Any other information on results incl. tables

Table 1. Concentrations of test substance in the test solutions.

Nominal concentration of test substance (mg/L)	Measured concentration of test substance (mg/L)
	0 h	72 h	Mean
Medium control	< 0.15^#1	< 0.15^#1	-
1.0	1.03 [103]	1.05 [105]	1.04 [104]
3.2	3.31 [103]	3.37 [105]	3.34 [104]
10	10.3 [103]	10.6 [106]	10.4 [104]
32	33.1 [103]	33.9 [106]	33.5 [105]
100	103 [103]	105 [105]	104 [104]

[ ] : percentage to the nominal concentration (%)

#1: less than quantification limit

Table 2. Percent growth inhibition (I_µ) for growth rate of P. subcapitata exposed to test substance.

	vessel n°	growth rate (µ/d)
Nominal concentration of test substance (mg/L)		0 - 72 h	Percent inhibition^#1 Iµ(%)
Medium control	1	1.5241	-
	2	1.5506	-
	3	1.5628	-
	4	1.6210	-
	5	1.5525	-
	6	1.6068	-
	Mean	1.5696	-
	SD	0.0369	-
	CV	2.3	-
1.0 [1.04]	1	1.5502	1.2
	2	1.5000	4.4
	3	1.5180	3.3
	Mean	1.5227	3.0
	SD	0.0255	-
3.2 [3.34]	1	1.5177	3.3
	2	1.5993	-1.9
	3	1.5986	-1.8
	Mean	1.5719	-0.1
	SD	0.0469	-
10 [10.4]	1	1.4246	9.2
	2	1.4579	7.1
	3	1.4912	5.0
	Mean	1.4579	7. 1†
	SD	0.0333	-
32 [33.5]	1	1.4401	8.3
	2	1.5487	1.3
	3	1.5057	4.1
	Mean	1.4981	4.6
	SD	0.0547	-
100 [104]	1	1.4509	7.6
	2	1.4373	8.4
	3	1.4487	7.7
	Mean	1.4456	7.9†
	SD	0.0073	-

[ ]: Mean measured concentration of test substance

†:Statistically different (Multiple comparison (Dunnett test, significance level=5%))

^#1: Percent inhibition relative to the medium control

SD: Standard deviation

CV: Coefficient of variation (%)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes